Targeted Mutagenesis of Mycobacterium Strains by Homologous Recombination

Targeted mutagenesis by homologous recombination (TMHR) is an efficient allelic exchange mutagenesis for bacterial genome engineering in synthetic biology. Unlike other allelic exchange methods, TMHR does not require a heterologous recombinase to insert or excise a selectable marker from the genome. In contrast, positive and negative selection is achieved solely by suicide vector-encoded functional and host cell proteins. Here we describe a concise protocol to knock out and knock in a 3-ketosteroid-1,2-dehydrogenase gene (kstd) in Mycobacterium neoaurum HGMS2 using TMHR approach. The homology arms flanking the kstd gene are amplified by PCR in vitro and then subcloned into a common homologous recombination vector. The vector is then electroporated into the HGMS2 competent cells. The replacement of the kstd gene by homologous recombination produces antibiotic-resistant single-crossover recombination via the first allelic exchange. Double-crossover markerless mutants are directly separated using sucrose-mediated counterselection. These two steps can generate seamless mutations down to a single DNA base pair. The whole process takes less than 2 weeks.